Project Summary/Abstract Defects in tissue stem cell self-renewal and differentiation are linked to organ malformations during fetal development and organ dysfunction in adult life. Despite this wide range of healthcare implications, the cellular and molecular regulation of stem cells is poorly understood, in particular with respect to the microenvironment in which tissue stem cells reside, termed the niche. An excellent model for studying stem cell biology is the mammalian spermatogonial stem cell (SSC) niche, as tight regulation of self-renewal and differentiation is required for the constant production of a large number of gametes over a long reproductive lifespan; imbalances in this process directly contribute to infertility or germ-cell-derived cancers. Despite research efforts, the cellular components of the SSC niche remain largely undefined. Our preliminary data reveal that macrophages are a critical part of the niche required for spermatogenesis. Macrophages are immune cells that are present throughout most organs and are traditionally associated with phagocytosis of foreign pathogens in the innate immune response; however, there is a growing awareness of tissue-specific developmental roles for macrophages. Additionally, several studies have reported that macrophages are located near tissue stem cell niches, but their roles in this biological context are unclear. Therefore, the mechanisms by which macrophages form part of stem cell niches and direct stem cell activity represent a significant knowledge gap in the field. In particular, specific roles for macrophages in the SSC niche have not been previously investigated. This research program will address important unanswered questions in SSC niche biology through three focused goals: 1) to identify the mechanisms used by macrophages to promote SSC differentiation; 2) to determine the signals that recruit macrophages to the stem cell niche; and 3) to define the role of macrophages in coordinating with other somatic cell types, such as Sertoli cells and peritubular myoid cells, to establish and maintain the niche. To accomplish these goals, we will employ: in vivo genetic mouse models; ex vivo culture systems; lineage-specific genomic studies of purified cell populations; and whole organ time-lapse live imaging. These approaches will allow us to obtain an in-depth knowledge of testis stem cell niche function that was previously unattainable. This work will identify novel cellular and molecular mechanisms directing the SSC niche and uncover new paradigms of stem cell biology that are relevant to both development and disease. Ultimately, this research will lead to new insights into the etiology of male infertility and testicular cancer, and aid in the creation of improved diagnostic and treatment methods for these conditions.